The primary goal of this proposal is to provide the applicant, James P. Hamilton, M.D., with a significant period of continued mentorship in a strong, supportive scientific environment in order for him to continue developing skills needed to become a fully independent physician-scientist. Dr. Hamilton has undergone superior clinical training in Gastroenterology, including a Fellowship in Advanced Hepatology. Moreover, during his career thus far, Dr. Hamilton has already focused his research interest on the molecular genetic basis of chronic liver disease. To advance his academic career as well as to establish his independence, further training is required in biochemistry, molecular biology, scientific study design, and data analysis. The research proposal described herein will investigate the expression and biologic functions of Thioredoxin Inhibitory Protein (TXNIP) in the pathogenesis of chronic liver disease. His hypothesis is that TXNIP activation in chronic hepatitis leads to increased reactive oxygen species (ROS), which in turn lead to liver fibrosis. He further postulates that once cirrhosis has developed, loss of TXNIP expression, due in part to hypermethylation, results in abnormal hepatocyte growth and proliferation. The proposed project will apply the considerable expertise and resources of the mentor's hepatic fibrosis research with the knowledge and experience of other key collaborators at Johns Hopkins. Specifically, Aim 1 of the current proposal will examine TXNIP expression at the mRNA and protein levels in normal, inflamed, cirrhotic and cancerous liver tissues, as well as in hepatocyte and hepatic stellate cell lines. Aim 2 will scrutinize the biological functions of TXNIP in chronic liver disease in vitro. TXNIP-induced generation of ROS and activation of pro-fibrotic pathways will be examined. Furthermore, effects of TXNIP on cell growth, proliferation, and apoptosis will be determined. Aim 3 will investigate the regulation of TXNIP by methylation. This mechanism will be evaluated directly, using bisulfite sequencing and methylation-specific PCR, as well as indirectly, using demethylating agents to reverse transcriptional silencing.